Breast cancer is the second leading cause of cancer death among women in the United States and is the leading cause of death among women aged 30-70. (Abeloff, M. D., Curr. Opin. Oncol., 8:447-448 (1996)). The inheritance of germ-line mutations in autosomal dominant susceptibility genes appears to be responsible for 5-10% of all breast cancer cases (Fitzgerald, M. G., et al., New Engl. J. Med., 334:143-149 (1996)), and up to 36% of the cases diagnosed before age 30. BRCA1 was the first isolated breast cancer susceptibility gene (Langston, A. A., et al., New Engl. J. Med., 334:137-142 (1996); Couch, F. J. and Weber, B. L., Hum. Mutat., 8:8-18 (1996)) and mutations in BRCA1 alone account for approximately 45% of the families with high incidence of breast and ovarian cancer (Chen, Y. M., et al., Science, 272:125-126 (1996); Sully, R., et al., Science, 272:123-126 (1996)). In addition, a second breast cancer susceptibility gene, BRCA2, has been isolated recently (Wooster, R., et al., Nature, 378:789-792 (1995); Tavtigian, S. V., et al., Nat. Genet., 12:333-337 (1996)).
However, the majority of breast carcinomas appear to be sporadic and have a complex accumulation of molecular and cellular abnormalities that constitute the malignant phenotype. A number of somatic gene alterations, such as loss of expression of specific tumor suppressor genes, have been found to occur in primary human breast tumors (Borg, A., et al., Cancer Res., 52:2991-2994 (1992); Eeles, R. A., et al., Cancer Surveys, 25:101-124 (1995)). Additionally, there is considerable evidence that genetic alterations in growth factor signaling pathways can contribute to human breast malignancies. In this regard, activation of different proto-oncogenes has been found in primary breast tumor (Berns, E. M., et al., Cancer Res., 52:1036-1039 (1992); Borg, A., et al., Brit. J. Cancer, 63:136-142 (1991); Gullick, W. J., et al., Brit. J. Cancer, 63:434-438 (1991)). Thus, there is considerable importance in identifying, at a molecular level, factors that contribute to the progression from normal growth towards malignancy.
The present invention relates to the demonstration that a cytoplasmic protein tyrosine kinase, Csk Homologous Kinase or CHK, is expressed in human breast cancer, but not in adjacent normal breast tissue. Specifically, the present invention relates to methods of detecting the presence of cancer in mammalian breast tissue by the detection of the protein tyrosine kinase CHK, or the detection of nucleic acids encoding the CHK in mammalian tissue, specifically breast tissue. The detection of CHK in breast tissue is indicative of cancer.
The presence of CHK in breast tissue can be determined by detecting the expression of CHK protein, or a protein fragment, in breast tissue samples obtained from the mammal. For example, biopsy tissue can be obtained from the mammal, fixed in a suitable medium and contacted with anti-CHK antibodies, for example rabbit anti-CHK, which specifically bind to the CHK protein if it is present in the tissue sample. The anti-CHK antibody can itself be detectably labeled, or a detectably labeled second antibody, for example, peroxidase-conjugated mouse anti-rabbit antibody, can be used.
The presence of CHK in breast tissue can also be detected using an immunoblot (e.g., Northern blot) assay. For example, tissue can be obtained from the mammal and a cell lysate prepared which contains proteins released from the tissue cells. The lysate proteins can be separated by electrophetic means, such as by size by SDS polyacrylamide gel electrophoresis, and contacted with anti-CHK antibody which specifically binds to CHK if it is present in the lysate. Again, the anti-CHK antibody can be detectably labeled, or a detectably labeled second antibody can be used. Alternatively, CHK protein present in a cell lysate can be detected by enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or other immunoassays.
The presence of CHK in breast tissue can also be determined by detecting the presence of a nucleic acid sequence encoding all, or a portion, of the CHK protein. The nucleic acid can be DNA or RNA. For example, genomic DNA, cDNA or RNA can be obtained from a sample of breast tissue and contacted with a polynucleotide (a nucleic acid probe) that forms a stable hybrid with the nucleic acid sequence encoding CHK. The probe can be detectably labeled. The DNA or RNA obtained from the mammal can be amplified prior to assay, for example using the polymerase chain reaction (PCR) or the ligase chain reaction (LCR), using specific nucleic acid primers. Primers useful to amplify the CHK nucleic acid specifically hybridize to the CHK nucleic acid or to nucleic acid sequence that flanks the target CHK nucleic acid sequence region.
Overexpression of the receptor tyrosine kinase, ErbB-2 (also termed neu/HER-2) has been associated with the development of breast cancer. (Slamon, D. J., et al., Science, 244:707-712 (1989); Olsson, H., et al., J. Natl. Cancer Instit., 83:1483-1487 (1991)). A common pathway linking the activation mechanisms in ErbB-2 amplification in breast cancer is increased tyrosine kinase activity which leads to cellular transformation. The abundance of ErbB-2 receptors and their ligands (e.g., heregulin or HGR) in breast cancer points to a functional role in the pathogenesis of this malignancy. As demonstrated herein, CHK specifically interacts with activated ErbB-2 upon HGR stimulation and results described herein suggest that CHK functions as a negative regulator of ErbB-2 mediated mitogenic signaling.
Accordingly, the present invention also encompasses methods of inhibiting breast cancer cell growth (also referred to herein as neoplastic cell growth), specifically ErbB-2 mediated neoplastic cell growth, by supplying CHK to cancer cells. For example, CHK protein, peptide or a biologically active fragment thereof, or a CHK analog or derivative, can be supplied to mammalian breast tissue which is abnormal, e.g., neoplastic, or at risk of becoming abnormal. The CHK protein can be supplied to the target breast tissue by introducing into target cells a liposome preparation that contains CHK. Specifically encompassed by this invention is the topical application of such liposomes in a cream or ointment.
Alternatively, CHK can be supplied to the target tissue by introducing a nucleic acid sequence encoding CHK, or a biologically active fragment, analog, or derivative of CHK which is then expressed in the breast tissue.
As described herein, for the first time, Csk-homologous Kinase has been identified as playing an important role in signaling in neoplastic breast tissue and as functioning as a negative regulator of ErbB-2. As a result of this work, novel methods of detecting and inhibiting breast cancer are now available.